Parenteral ceftolozane antibiotic compositions

ABSTRACT

Parenteral pharmaceutical compositions including ceftolozane and tazobactam can be formulated to provide desired levels of osmolality, pH and ceftolozane stability.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/792,092, filed Mar. 15, 2013; U.S. Provisional Application No. 61/793,007, filed Mar. 15, 2013; U.S. Provisional Application No. 61/882,936, filed Sep. 26, 2013; and U.S. Provisional Application No. 61/893,436, filed Oct. 21, 2013. The contents of these applications are incorporated hereby by reference in their entirety.

TECHNICAL FIELD

This application discloses parenteral pharmaceutical compositions comprising ceftolozane.

BACKGROUND

Increasing resistance to commonly prescribed antimicrobial agents remains a serious global problem emphasizing the need for new antimicrobial agents to treat infections caused by difficult-to-treat pathogens such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The CDC reports that antibiotic resistance is one of the most serious health threats today. The CDC estimates that antibiotic resistant infections affect over 2 million people in the US annually with an estimated 23,000 people dying as a direct result. There remains an unmet medical need for new antibiotic therapies to treat gram-negative pathogens.

Studies have demonstrated that ceftolozane/tazobactam has a broad spectrum of activity against Gram-negative bacteria. Ceftolozane is a cephalosporin antibacterial agent. Ceftolozane is also referred to as CXA-101, FR264205, (6R,7R)-3-[(5-amino-4-{[(2-aminoethyl)carbamoyl]amino}-1-methyl-1H-pyrazol-2-ium-2-yl)methyl]-7-({(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(1-carboxy-1-methylethoxy)imino]acetyl}amino)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, or (6R,7R)-3-[5-Amino-4-[3-(2-aminoethyl)ureido]-1-methyl-1H-pyrazol-2-ium-2-ylmethyl]-7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(Z)-1-carboxy-1-methylethoxyimino]acetamido]-3-cephem-4-carboxylic acid). Ceftolozane sulfate is a pharmaceutically acceptable ceftolozane salt of formula (I) that can be formulated for intravenous administration or infusion.

The antibacterial activity of ceftolozane is believed to result from its interaction with penicillin binding proteins (PBPs) to inhibit the biosynthesis of the bacterial cell wall which acts to stop bacterial replication. CXA-201 refers to a combination of ceftolozane and the β-lactamase inhibitor (“BLI”) tazobactam with a 2:1 weight ratio between the amount of ceftolozane active and tazobactam active. Tazobactam is a BLI against Class A and some Class C β-lactamases. CXA-201 is being evaluated as a parenteral antibiotic for the treatment of infections caused by bacterial pathogens such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, including in the treatment of complicated urinary tract infections and complicated intra-abdominal infections. Tazobactam potentiates the activity of ceftolozane against Acinetobacter spp. and common species of Enterobacteriaceae, including Citrobacter spp., Enterobacter cloacae, E. coli, K. pneumoniae, Proteus mirabilis, and Serratia marcescens. These surveillance data demonstrate that 88% to 100% of these Enterobacteriaceae species are inhibited at <8 μg/mL. Tazobactam had little impact on the susceptibility of P. aeruginosa to ceftolozane.

The stability ceftolozane in certain lyophilized pharmaceutical compositions initially evaluated for intravenous administration was improved by lyophilizing a solution of ceftolozane sulfate with a stabilizing-effective amount of sodium chloride, sucrose or trehalose (e.g., 125 mg-500 mg of sodium chloride or about 300 mg of sucrose or about 300 mg of trehalose per 1,000 mg of ceftolozane active) and an amount of an alkalizing agent such as L-arginine providing a pH of about 5-7 in the solution (e.g., about 600 mg L-arginine per 1,000 mg ceftolozane active).

For example, an improvement in ceftolozane purity was observed by high performance liquid chromatography (HPLC) after stability testing over 7 days at 60 degrees C. (e.g., Examples below) in ceftolozane compositions obtained by lyophilizing aqueous solutions of ceftolozane sulfate with stabilizing-effective amounts of sodium chloride at a pH of 5-7.

Although increased concentrations of sodium chloride and L-arginine stabilized lyophilized ceftolozane compositions, these additional components also raise the osmolality of resulting pharmaceutical compositions upon reconstitution. Osmolality is a measure of the number of dissolved particles per unit weight (mOsm/kg) of a parenteral pharmaceutical composition. This value can be calculated or determined experimentally by osmometry. Normal plasma has an osmolality in the range of 285-295 mOsm/kg (osmolarity range of 300-310 mOsm/L). Agents with an undesirably high osmolality can cause pain and/or other negative physiologic disturbances (e.g., excessively high osmolality can cause red blood cell crenation upon intravenous injection and/or phlebitis while intravenous administration of agents with excessively low can cause hemolysis). Accordingly, there is a need for parenteral formulations of ceftolozane/tazobactam for injection having both a desired ceftolozane chemical stability and a physiologically and therapeutically appropriate osmolality (e.g., less than 800 mOsmol/kg and preferably less than 600 mOsmol/kg) and pH (e.g., 5-7) when prepared in a pharmaceutically acceptable carrier for administration to a patient in need thereof.

There remains a need for ceftolozane/tazobactam injectable pharmaceutical compositions formulated for parenteral administration that provide desired levels of ceftolozane stability in an injectable form (e.g., a stabilizing-effective amount of sodium chloride) comprising a BLI (e.g., tazobactam), while remaining compatible with relevant biological characteristics including a providing pharmaceutically acceptable and therapeutically appropriate levels of pH and osmolality.

SUMMARY

Parenteral pharmaceutical compositions can be obtained by dissolving a solid unit dosage form containing ceftolozane in a stabilized solid form into one or more pharmaceutically acceptable liquid carriers selected from the group consisting of water for injection, 0.9% sodium chloride aqueous solution for injection, 0.45% sodium chloride aqueous solution for injection, and 5% dextrose for injection and water for injection. Ceftolozane can be stabilized in a solid form (e.g., a lyophilized powder) pharmaceutical composition by combination with stabilizing-effective amounts of sodium chloride and L-arginine. The solid form pharmaceutical composition is preferably formulated for parenteral administration for the treatment of infections, comprising a therapeutically effective amount of ceftolozane sulfate and tazobactam in a ratio of 1,000 mg ceftolozane active per 500 mg of tazobactam active, in addition to excipients selected and provided in amounts effective to stabilize ceftolozane in the solid form pharmaceutical composition. The invention is based in part on the discovery that therapeutically effective doses of solid form stabilized ceftolozane compositions can form injectable liquid pharmaceutical compositions having osmolality levels of about 600 mOsm/kg or less, despite increased levels of ceftolozane-stabilizing excipients such as sodium chloride and L-arginine, solid form pharmaceutical compositions can be reconstituted in isotonic and even certain hypertonic diluents and carriers.

A parenteral ceftolozane/tazobactam pharmaceutical antibiotic composition having an osmolality of less than 800 mOsmol/kg (i.e., 400-500 mOsm/kg) at a pH of about 5-7 includes an amount of ceftolozane sulfate providing 1,000 mg ceftolozane active, about 125 mg to 500 mg sodium chloride per 1,000 mg ceftolozane active, and an amount of tazobactam sodium providing 500 mg of tazobactam per 1,000 mg ceftolozane active at a pH of 5-7 in a pharmaceutically acceptable carrier. The parenteral pharmaceutical antibiotic composition can have an osmolality of 400-500 mOsm/kg at a pH of 5-7, the composition comprising an amount of ceftolozane sulfate providing 1,000 mg ceftolozane active, about 450 mg to 500 mg sodium chloride per 1,000 mg ceftolozane active, and an amount of tazobactam sodium providing 500 mg of tazobactam free acid, provided in one or more pharmaceutically acceptable liquid carriers selected from the group consisting of water for injection, 0.9% sodium chloride aqueous solution for injection, 0.45% sodium chloride aqueous solution for injection, and 5% dextrose for injection and water for injection. The composition can be a solution for injection having a total volume of about 110 mL and can have one or more of the following characteristics in any combination: contains a total of about 1,147 mg ceftolozane sulfate; includes L-arginine in an amount effective to provide a pH of about 5-7, includes about 600 mg of L-arginine per 1,000 mg of ceftolozane active; includes citric acid (e.g., 21 mg per 1,000 mg of ceftolozane active). The composition also include a pharmaceutically acceptable carrier that can be 0.9% sodium chloride aqueous solution for injection, and can be obtained by dissolving a composition having the vial composition in Table 1a below.

TABLE 1a Solid Form Antibacterial Pharmaceutical Composition Nominal Composition Component mg per Vial Ceftolozane Sulfate about 1147 (providing 1,000 mg ceftolozane active) Citric Acid  21 Sodium Chloride 487 L-Arginine 600 Q.S. for pH adjustment Tazobactam about 537 (providing 500 mg tazobactam free acid Sodium per 1,000 mg ceftolozane active) Nitrogen Q.S. A parenteral pharmaceutical antibiotic composition can be obtained by a process further comprising reconstituting the unit dosage form in the vial using 0.9% sodium chloride aqueous solution for injection.

Methods of treating a bacterial infection in a subject comprising can include administering to a patient in need thereof a parenteral pharmaceutical antibiotic composition having an osmolality of 400-500 mOsm/kg at a pH of 5-7, the composition comprising an amount of ceftolozane sulfate providing 1,000 mg ceftolozane active, about 450 mg to 500 mg sodium chloride per 1,000 mg ceftolozane active, and an amount of tazobactam sodium providing 500 mg of tazobactam free acid; where the parenteral antibiotic composition also includes a pharmaceutically acceptable liquid carrier selected from the group consisting of 0.9% sodium chloride aqueous solution for injection, 5% dextrose for injection and water for injection. The parenteral pharmaceutical antibiotic composition can be intravenously administered to treat a bacterial infection that is a complicated urinary tract infection, a complicated intra-abdominal infection, and/or is caused by bacterial pathogens selected from the group consisting of: Staphylococcus aureus, Escherichia coli, Acinetobacter baumanii, Haemophilus influenzae, Klebsiella pneumonia, and Pseudomonas aeruginosa. The method can include treating a bacterial infection by steps including reconstituting a unit dosage form of ceftolozane/tazobactam for injection according having the composition in Table 1 in a pharmaceutically acceptable liquid carrier to obtain a reconstituted antibiotic composition; and intravenously administering the reconstituted antibiotic composition within the parenteral pharmaceutical antibiotic composition. The method of treatment can also include comprising repeatedly intravenously administering separate doses of the parenteral pharmaceutical antibiotic composition to a patient in need thereof three times per day. Each separate parenteral pharmaceutical antibiotic compositions can be administered to the patient over a 1 hour period.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a reference HPLC chromatogram showing the peaks of ceftolozane (CXA-101) and related additional compounds.

FIG. 2 is a plot of the data points from Table 5, showing the purity of the ceftolozane in CXA-101 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by the HPLC method described in Example 5, wherein the CXA-101 compositions comprise ceftolozane and sodium chloride.

FIG. 3 is a plot of the data points from Table 6, showing the peak area of the composition peak 1 in CXA-101 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by the HPLC method described in Example 5, wherein the CXA-101 compositions comprise ceftolozane and sodium chloride.

FIG. 4 is a plot of the data points from Table 7, showing the total peak area of the composition with a RRT of 0.43 and the composition peak 3 in CXA-101 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by the HPLC method described in Example 5, wherein the CXA-101 compositions comprise ceftolozane and sodium chloride.

FIG. 5 is a plot of the data points from Table 8, showing the peak area of the composition peak 7 in CXA-101 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by the HPLC method described in Example 5, wherein the CXA-101 compositions comprise ceftolozane and sodium chloride.

FIG. 6 is a plot of the data points from Table 13, showing the purity of ceftolozane in CXA-201 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by the HPLC method described in Example 5, wherein the CXA-201 compositions comprise ceftolozane, tazobactam, and sodium chloride.

FIG. 7 is a plot of the data points from Table 14, showing the peak area of the composition peak 1 in CXA-201 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by the HPLC method described in Example 5, wherein the CXA-201 compositions comprise ceftolozane, tazobactam, and sodium chloride.

FIG. 8 is a plot of the data points from Table 15, showing the total peak area of the composition with a RRT of 0.43 and the composition peak 3 in CXA-201 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by the HPLC method described in Example 5, wherein the CXA-201 compositions comprise ceftolozane, tazobactam, and sodium chloride.

FIG. 9 is a plot of the data points from Table 16, showing the peak area of the composition peak 7 in CXA-201 compositions at 60° C. on day 0, day 1, day 3, and day 7, as measured by the HPLC method described in Example 5, wherein the CXA-201 compositions comprise ceftolozane, tazobactam, and sodium chloride.

DETAILED DESCRIPTION

Solid form ceftolozane compositions can be prepared with components selected and provided in ceftolozane stabilizing-effective amounts. These solid form compositions can be dissolved in various pharmaceutically acceptable carriers to obtain aqueous parenteral pharmaceutical compositions having a suitable pH and osmolality for intravenous administration.

In particular, ceftolozane can be stabilized in solid form compositions (e.g., lyophilized ceftolozane compositions) by lyophilizing an aqueous solution including a stabilizing effective amount of sodium chloride (e.g., 125 mg to 500 mg sodium chloride per 1,000 mg ceftolozane active, preferably 450 mg to 500 mg sodium chloride per 1,000 mg ceftolozane active) and L-arginine (e.g., 500-700 mg L-arginine per 1,000 mg of ceftolozane active, preferably 600 mg L-arginine per 1,000 mg of ceftolozane active). Unit dosage forms of these lyophilized solid form ceftolozane compositions containing therapeutically effective doses of ceftolozane can be blended with tazobactam (e.g., in amounts of active and excipients providing 1,000 mg of sufficiently stabilized ceftolozane active and 500 mg tazobactam acid for treatment of certain urinary tract infections or certain intra-abdominal infections, or 2,000 mg ceftolozane active and 1,000 mg of tazobactam acid for certain pneumonia infections). The unit dosage forms can be dissolved at a pH of about 5-7 to obtain aqueous parenteral pharmaceutical compositions with an osmolality that is less than about 600 mOsmol/kg (e.g., about 400-600) upon reconstitution for intravenous administration to a subject.

Parenteral Pharmaceutical Compositions

The parenteral pharmaceutical compositions can be obtained by dissolving and diluting a solid form pharmaceutical compositions such as the unit dosage form in Table 1 (Example 1). Typically, the unit dosage form (e.g., a vial or bag) can be dissolved in a small volume of a pharmaceutically acceptable diluent (e.g., 10-20 mL of water for injection), withdrawn from the unit dosage form container, and then diluted to a larger injection volume in the same or a different pharmaceutically acceptable carrier (e.g., 0.9% normal aqueous saline for injection) to form the parenteral pharmaceutical composition. A number of different scenarios can be used in the preparation of a parenteral pharmaceutical composition from a unit dosage form, including the use of pharmaceutically acceptable diluents and carriers independently selected from the group consisting of: water for injection, 0.9% and 0.45% sodium chloride aqueous solutions for injection, 5% dextrose for injection, and combinations thereof (e.g., Example 3). The diluent and carrier, and composition of the parenteral pharmaceutical composition can be selected to provide a suitable osmolality, pH, and a therapeutically effective dose of the pharmaceutically active component(s) in a liquid parenteral pharmaceutical composition.

Solid Form Pharmaceutical Compositions

As described herein (e.g., Examples 4a and 4b), adding stabilizing effective amounts of sodium chloride to compositions comprising ceftolozane (e.g., 125-500 mg, preferably about 450-500 mg sodium chloride per 1000 mg of ceftolozane) can stabilize ceftolozane in a solid form of a pharmaceutical composition (e.g., a lyophilized powder). Including stabilizing-effective amounts of sodium chloride can reduce the rate of formation of certain additional ceftolozane related substances when compared to samples with a lower sodium chloride concentration. Further, adding about 487 mg sodium chloride per 1000 mg of ceftolozane to a ceftolozane/tazobactam (e.g., CXA-201) composition can also inhibit the formation of certain additional compounds. For example, in one experiment, CXA-201 compositions comprising 125-481 mg sodium chloride per 1000 mg ceftolozane developed a reduced amount of a composition having a retention time of 63 minutes (“RT 63′”) after three months at 25° C. (see the HPLC measurements shown in, Table 11).

Methods of Treatment

The spectrum of activity for ceftolozane/tazobactam includes many clinically relevant, Gram negative pathogens including members of the Enterobacteriaceae such as E. coli and K. pneumoniae, nonfermenters such as P. aeruginosa, Gram-positive pathogens such as Streptococcus pneumoniae and S. pyogenes, and anaerobic pathogens such as Bacteroides fragilis (M2.6.2.4). Ceftolozane has stability to common cephalosporin-resistance mechanisms, including penicillinases, cephalosporinases, most common class A β-lactamases, AmpC hyperexpression and efflux mechanisms, and is little affected by porin deficiency. Ceftolozane/tazobactam compositions are active against strains of P. aeruginosa that are resistant to carbapenems, cephalosporins, fluoroquinolones and/or aminoglycosides, including many MDR isolates. The minimum inhibitory concentration for 90% of strains (MIC90) for ceftolozane/tazobactam against a wide array of P. aeruginosa strains (MIC90 0.5/4 μg/mL) is the lowest among all systemically administered antipseudomonal antibiotics, except for colistin. Ceftolozane/tazobactam is active against the majority of Enterobacteriaceae. The minimum inhibitory concentration (MIC) for 50%/90% of strains (MIC50/90) for E. coli is 0.25/0.5 μg/mL and for E. coli strains with an ESBL phenotype the MIC50/90 is 0.5/4 μg/mL.

Non-limiting examples of the bacterial infections that can be treated by the methods of the invention include infections caused by: aerobic and facultative gram-positive microorganisms (e.g., Staphylococcus aureus, Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pneumonia, Streptococcus pyogenes, Viridans group streptococci), aerobic and facultative gram-negative microorganisms (e.g., Acinetobacter baumanii, Escherichia coli, Haemophilus influenza, Klebsiella pneumonia, Pseudomonas aeruginosa, Citrobacter koseri, Moraxella catarrhalis, Morganella morganii, Neisseria gonorrhoeae, Proteus mirabilis, Proteus vulgaris, Serratia marcescens, Providencia stuartii, Providencia rettgeri, Salmonella enterica), gram-positive anaerobes (Clostridium perfringens), and gram-negative anaerobes (e.g., Bacteroides fragilis group (e.g., B. fragilis, B. ovatus, B. thetaiotaomicron, and B. vulgates), Bacteroides distasonis, Prevotella melaminogenica).

In one aspect, provided herein is a method for treating a bacterial infection in a subject in need thereof comprising intravenously administering to the subject a therapeutically effective amount of a liquid pharmaceutical formulation comprising ceftolozane, tazobactam, and 125-500 mg sodium chloride per 1000 mg ceftolozane active, wherein the liquid pharmaceutical composition has an osmolality between about 350-900 mOsm/kg (including, e.g., 400-600 mOsm/kg) and a neutral pH.

In another aspect, provided herein is a method for treating a complicated intra-abdominal infection (cIAI) or a complicated urinary tract infection (cUTI) in a subject in need thereof, comprising intravenously administering to the subject a liquid pharmaceutical formulation comprising 1000 mg ceftolozane active, 500 mg of tazobactam active, and 125-500 mg of sodium chloride per 1000 mg ceftolozane active, wherein the liquid pharmaceutical formulation has an osmolality between about 450 mOsm/kg and 900 mOsm/kg and a neutral pH. In a further embodiment of the methods provided herein, the liquid pharmaceutical formulation has an osmolality less than about 600 mOsm/kg.

In yet another aspect, provided herein is a method for treating nosocomial pneumonia in a subject in need thereof, comprising intravenously administering to the subject a liquid pharmaceutical formulation comprising 2000 mg ceftolozane active, 1000 mg of tazobactam active, and 125-500 mg of sodium chloride per 1,000 mg ceftolozane, wherein the liquid pharmaceutical formulation has an osmolality between about 450 mOsm/kg and 900 mOsm/kg and a neutral pH. In a further embodiment of the methods provided herein, the liquid pharmaceutical formulation has an osmolality between about 450 mOsm/kg and 600 mOsm/kg.

In an embodiment of the methods described herein, the liquid pharmaceutical formulation (e.g., an intravenous infusion solution) is intravenously administered over a 60 minute period, 3 times per day (e.g., every 8 hours).

In another embodiment of the methods described herein, the method further comprising the steps of reconstituting a solid composition of ceftolozane, tazobactam, and sodium chloride to produce an intravenous infusion solution, wherein the solution has an osmolality between about 600-900 mOsm/kg and more preferably 400-600 mOsm/kg, and administering a therapeutically effective amount of the solution to said subject.

In a further embodiment, an infusion solution comprising a unit dosage form of ceftolozane/tazobactam for injection is prepared via reconstitution and/or dilution with 0.9% Sodium Chloride Injection USP and 5% Dextrose Injection USP, which are both commonly used for reconstituting and diluting parenteral formulations. Sodium Chloride Injection, USP 0.9%, is a sterile, nonpyrogenic, isotonic solution. Each milliliter contains 9 mg sodium chloride in 9 mg injection. In another embodiment, the infusion solution is prepared via reconstitution and/or dilution with 0.5% Dextrose Injection, USP solution is sterile and nonpyrogenic parenteral solution containing dextrose in water for injection and is intended for intravenous administration. Each 100 mL of 5% Dextrose Injection, USP, contains dextrose, hydrous 5 g in water for injection. The caloric value is 170 kcal/L. The osmolality is 252 mOsmol/L (calc.), which is slightly hypotonic. The solution pH is 4.3 (3.2 to 6.5). In yet another embodiment, the infusion solution is reconstituted or diluted in part by sterile water or water for injection (WFI).

As used herein, a “neutral pH” refers to a pH of about 6.0 to about 8.0. In certain embodiments, a “neutral pH” refers to a pH that is approximately 7.0. In other embodiments, a “neutral pH” refers to a pH of about 6.0 to 7.5 or 6.0 to about 7.0. In yet another embodiment, a “neutral pH” refers to a pH of about 7.4.

As used herein, “osmolality” refers to the concentration of particles dissolved in solution expressed as osmoles of solute per kilogram of solvent. Human blood, for example, has an osmolality of about 290 mOsm/L, which corresponds to a 290×10³ M concentration of dissolved particles. Marc Stranz, A Review of pH and Osmolarity, 6 Int'l J. of Pharm. Compounding 216, 218 (May/June 2002) This value can be calculated or determined experimentally by osmometry (see, e.g., Example 3). In certain embodiments, the osmolality is between about 450 mOsm/kg and 900 mOsm/kg. In other embodiments, the liquid pharmaceutical formulation (e.g., an intravenous infusion solution) has an osmolality that is between about 450 mOsm/kg and 600 mOsm/kg. In preferred embodiments, the liquid pharmaceutical formulation (e.g., an intravenous infusion solution) has an osmolality that is less than about 600 mOsm/kg.

Ceftolozane is the cephalosporin (6R,7R)-3-[(5-amino-4-{[(2-aminoethyl)carbamoyl]amino}-1-methyl-1H-pyrazol-2-ium-2-yl)methyl]-7-({(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(1-carboxy-1-methylethoxy)imino]acetyl}amino)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate, and is also referred to as “CXA-101,” FR264205, or (6R,7R)-3-[5-Amino-4-[3-(2-aminoethyl)ureido]-1-methyl-1H-pyrazol-2-ium-2-ylmethyl]-7-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(Z)-1-carboxy-1-methylethoxyimino]acetamido]-3-cephem-4-carboxylic acid). “Ceftolozane” includes both ceftolozane free base and salts of ceftolozane, such as ceftolozane sulfate. Ceftolozane sulfate is a pharmaceutically acceptable salt of ceftolozane that can be combined with sodium chloride and other components to obtain an antibiotic composition suitable for administration by injection or infusion.

Unless otherwise indicated herein, the phrase “1000 mg ceftolozane” or “1 g ceftolozane” refers to an amount of ceftolozane containing the free base equivalent weight of ceftolozane provided in any suitable salt form. For example, a composition containing 1000 mg of ceftolozane in the ceftolozane sulfate solid form will include greater than 1000 mg of material (e.g., due to at least the additional weight of the sulfate counter ion). Preferably, a composition containing “1000 mg of ceftolozane” includes an amount of ceftolozane sulfate comprising 1000 mg of the ceftolozane molecule in free base equivalent form. For example, as shown in Table 1, 1147 mg ceftolozane sulfate corresponds to 1000 mg of ceftolozane free base.

In an embodiment, ceftolozane can be ceftolozane drug product intermediate (composition) wherein the ceftolozane composition further comprises excipients, stabilizers, or pH adjusting additives. For example, the ceftolozane can include sodium chloride, L-arginine, and citric acid.

The disclosed pharmaceutical compositions can include a β-lactamase inhibitor, such as tazobactam (CAS#: 89786-04-9), avibactam (CAS#1192500-31-4), Sulbactam (CAS#68373-14-8) and/or clavulanate (CAS#58001-44-8). The beta lactamase inhibitor can be included in a crystalline or amorpous form, such as a lyophilized tazobactam or crystalline tazobactam (e.g., U.S. Pat. Nos. 8,476,425 and 5,763,603) to obtain the pharmaceutical composition. Tazobactam is the most commonly used BLI in the disclosed pharmaceutical compositions. It is typically used as the free acid, sodium salt or arginine salt. Tazobactam free acid has the following structure:

“Ceftolozane active” refers to active portion of a salt form of ceftolozane, i.e., the free base form of ceftolozane. “Tazobactam active” refers to the active portion of a salt form of tazobactam, i.e., tazobactam free acid. The disclosed pharmaceutical compositions commonly have a 2:1 w/w ratio ceftolozane active to tazobactam active. A unit dosage form of the disclosed pharmaceutical composition typically has 1000 mg of ceftolozane active, preferably from ceftolozane sulfate and 500 mg of tazobactam active, preferably from tazobactam free acid, sodium salt or arginine salt. Another unit dosage form of the disclosed pharmaceutical composition has 2000 mg ceftolozane active, preferably from ceftolozane sulfate and 1000 mg of tazobactam active, preferably from tazobactam free acid, sodium salt or arginine salt.

As used herein, “treating”, “treat” or “treatment” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a pharmaceutical composition of the present invention to alleviate the symptoms or complications of a disease, condition or disorder, or to eliminate the disease, condition or disorder. The term “treat” can also include treatment of a cell in vitro or an animal model.

By a “therapeutically effective amount” of a compound of the invention is meant a sufficient amount of the compound to treat the disorder (e.g., bacterial infection). The specific therapeutically effective amount that is required for the treatment of any particular patient, subject, or organism (e.g., a mammal) will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound or composition employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see, for example, Goodman and Gilman's, “The Pharmacological Basis of Therapeutics”, Tenth Edition, A. Gilman, J. Hardman and L. Limbird, eds., McGraw-Hill Press, 155-173, 2001, which is incorporated herein by reference in its entirety). The therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.

In one embodiment, provided herein is a method for the treatment of an infection in a subject, where the infection is caused by Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, or Streptococcus pneumoniae comprising administering to said subject a therapeutically effective amount of a liquid pharmaceutical formulation having an osmolality between about 450 mOsm/kg and 900 mOsm/kg and a pH of about 6.0-8.0 comprising approximately 1147 mg ceftolozane sulfate; approximately 537 mg tazobactam sodium; and 400-500 mg sodium chloride. In one embodiment, the pharmaceutical composition further comprises 500-650 mg L-arginine and 15-30 mg anhydrous citric acid.

In another embodiment, provided herein is a method for the treatment of urinary tract infection, intra-abdominal infection, or nosocomial pneumonia in a subject, comprising administering to said subject a therapeutically effective amount of a liquid pharmaceutical formulation having an osmolality between about 450 mOsm/kg and 900 mOsm/kg and a pH of about 6.0-8.0 comprising approximately 1147 mg ceftolozane sulfate; approximately 537 mg tazobactam sodium; and 400-500 mg sodium chloride. In an embodiment, the pharmaceutical formulation comprises 487 mg sodium chloride. In one embodiment, the pharmaceutical formulation further comprises 500-650 mg L-arginine and 15-30 mg anhydrous citric acid.

In one embodiment, provided herein is a method for the treatment of an infection in a subject, wherein the infection is caused by Pseudomonas aeruginosa, Serratia marcescens, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, or Streptococcus pneumoniae comprising administering to said subject a therapeutically effective amount of a liquid pharmaceutical composition having an osmolality between about 450 mOsm/kg and 900 mOsm/kg and a pH of about 6.0-8.0 comprising approximately 1147 mg ceftolozane sulfate; approximately 537 mg tazobactam sodium; approximately 487 mg sodium chloride; and approximately 600 mg L-arginine. In one embodiment, the pharmaceutical formulation further comprises 15-30 mg anhydrous citric acid.

In another embodiment, provided herein is a method for the treatment of urinary tract infection, intra-abdominal infection, or nosocomial pneumonia in a subject, comprising administering to said subject a therapeutically effective amount of a liquid pharmaceutical formulation having an osmolality between about 450 mOsm/kg and 900 mOsm/kg and a pH of about 6.0-8.0 comprising approximately 1147 mg ceftolozane sulfate; approximately 537 mg tazobactam sodium; approximately 487 mg sodium chloride; and approximately 600 mg L-arginine. In one embodiment, the pharmaceutical formulation further comprises 15-30 mg anhydrous citric acid.

Any patent, patent application, publication, or other disclosure material identified in the specification is hereby incorporated by reference herein in its entirety. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material.

Unless otherwise indicated, as used herein, the term “Related Substances” with respect to HPLC detection refers to all the ceftolozane related process impurities and degradation products other than ceftolozane separated and detected by HPLC according to Example 5. Unless otherwise indicated, as used herein, the term “% Related Substances” refers to the % of the total HPLC peak area obtained by Example 5 attributed to all the ceftolozane related process impurities and degradation products other than ceftolozane.

ILLUSTRATIVE EXAMPLES OF SELECTED EMBODIMENTS OF THE INVENTION Example 1 Physicochemical and Biological Properties Ceftolozane/Tazobactam for Injection, 1000 mg/500 mg

As a product intended for intravenous use, several properties are important for physiological compatibility. These include particulate matter, sterility, endotoxin limit, pH, and osmolality. Particulate matter and sterility are controlled at the point of manufacture. The drug product is processed aseptically throughout the entire manufacturing process, inclusive of ceftolozane, tazobactam sodium, and ceftolozane/tazobactam in-vial drug product.

The ceftolozane/tazobactam drug product is controlled to approximately pH 6, to provide physiological comfort, while still assuring adequate stability for the drug substances. The ceftolozane drug product intermediate is controlled during compounding to pH 6.5±0.5 and is preferably pH 5 to 7. The tazobactam sodium is preferably at pH 5 to 7.

Ceftolozane/tazobactam following reconstitution with normal saline and dilution for infusion also in normal saline (10 mg/mL ceftolozane; 5 mg/mL tazobactam) is slightly hypertonic, with osmolality approximately 500 mOsm/kg. However, slightly hypertonic intravenous infusion solutions are not uncommon as drug products are commonly prepared and diluted with already-isotonic solutions, such as normal saline. The generally accepted maximum upper limit for peripheral intravenous administration is approximately 900 mOsm/kg, though admixtures 600 to 900 mOsm/kg are typically administered through a central line. Therefore, to be within the limits of this range, the infusion product is preferably less than 600 mOsm/kg. An example of a unit composition of a dosage for reconstitution is described in Table 1.

TABLE 1 Unit Compositions of Ceftolozane/Tazobactam for Injection, 1000 mg/500 mg Nominal Composition Component Function mg per Vial Ceftolozane Ceftolozane Active 1147 composition¹⁾ Sulfate Citric Acid, Chelating Agent 21 Anhydrous Sodium Stabilizing Agent 487 Chloride L-Arginine Alkalizing Agent 600²⁾ Q.S. for pH adjustment Tazobactam Sodium³⁾ Active 537 Nitrogen Processing Aid^((a)) Q.S. Total Weight 2792 ¹⁾Actual amount of Ceftolozane composition will vary based on the measured potency. Ceftolozane sulfate, 1147 mg, corresponds to 1000 mg Ceftolozane free base. ²⁾L-arginine is added as needed to achieve pH 6.5 ± 0.5; 600 mg per vial is considered a representative total amount. ³⁾Actual weight of tazobactam sodium will vary based on the measured potency. Tazobactam sodium 537 mg, corresponds to 500 mg tazobactam free acid ⁴⁾Nitrogen blanket is applied after powders are dispensed to the vial and prior to insertion of stopper.

Example 2 Excipients in Ceftolozane Drug Product Intermediate

The excipients in ceftolozane composition were chosen to ensure stability and processability of the ceftolozane drug substance into the drug product. The specific excipients, their quantities and functions are provided in Table 2. All excipients are compendial and typical for sterile pharmaceutical dosage forms, requiring no additional treatment prior to use in the formulation. The excipients are used in levels within the range established in other FDA approved products as described in the Inactive Ingredients Database (IID).

TABLE 2 Excipients Used in Ceftolozane composition Concentration Inactive Ingredients Amount, in Infusion Rationale for Database Component Function mg/Vial Solution, % Inclusion (IID) Range Citric acid Chelating 21 0.02 Used to prevent 0.0025 to 50% agent discoloration and degradation Sodium Stabilizing 487 0.49 Used as a stabilizing 0.187 to 45% Chloride agent agent for ceftolozane sulfate L-arginine Alkalizing 600^((a)) Q.S. 0.60 Used to adjust 0.29 to 88% agent for pH ceftolozane solution adjustment pH ^((a))L-arginine is added as needed to achieve pH 6.5 ± 0.5; 600 mg per vial is considered a representative total amount.

Example 3 Determining Osmolality of CXA-201Compositions Example 3a Osmolality for an Injectable Pharmaceutical Composition Comprising the Composition of Table 1

The osmolality data for injectable pharmaceutical compositions in Table 3a corresponds to the osmolality of unit dosage forms of ceftolozane/tazobactam for injection formulated by reconstituting the sample described in Table 1 with various diluents listed below, then injecting the reconstituted solution into a 100 mL bag of an injectable pharmaceutically acceptable carrier. Data for osmolality of the following product reconstitution scenarios is shown in Table 3a, as determined using the composition from Table 1 and the corresponding diluent and injectable carrier as indicated herein.

-   -   5% Dextrose Injection USP, 100 mL Bag (Baxter)     -   0.9% Sodium Chloride Injection USP, 100 mL Bag (Baxter)     -   sWFI-D5W: reconstituted with Sterile WFI then added into 5%         Dextrose Injection bag     -   NS-D5W: reconstituted with USP Normal Saline then added into 5%         Dextrose Injection bag     -   sWFI-NS: reconstituted with Sterile WFI then added into 0.9%         Sodium Chloride Injection Bag     -   NS-NS: reconstituted with USP Normal Saline then added into 0.9%         Sodium Chloride Injection bag

TABLE 3a Osmolality of Ceftolozane Bag Solution (mOsm/kg) Time Point sWFI - D5W NS - D5W sWFI - NS NS - NS RT T0 446 470 449 478

Example 3b Osmolality for Other Injectable Pharmaceutical Compositions

Additional Ceftolozane and Tazobactam Sodium samples (described below as #1-#3) were reconstituted as follows:

-   Sample#1: Weighed 0.103 g of Tazobactam Sodium and 0.462 g of     CXA-101dissolved in 4 mL of WFI Water and 6 mL of USP Normal Saline. -   Sample#2: Weighed 0.103 g of Tazobactam Sodium and 0.462 g of     CXA-101dissolved in 4 mL of WFI Water added 10 mL of USP Normal     Saline. -   Sample#3: Weighed 0.103 g of Tazobactam Sodium dissolved in 1 mL of     WFI Water and 0.462 g of CXA-101dissolved in 1 mL of WFI Water then     mixed together added 10 mL of USP Normal Saline. -   Tazobactam Sodium (Potency: 97.5%) -   CXA-101 (Potency: 43.3%)

WFI Water USP Normal Saline

The osmolality of CXA-101 and Tazobactam Sodium samples (#1-#3) was then determined using a freezing point depression Osmometer (available from Advanced Instruments, Inc.).

TABLE 3b Osmolality of Reconstituted Solutions CXA-101 Tazobactam Conc. Sodium Osmolality Sample# (mg/mL) Conc. (mg/mL) WFI Water Saline (mOsm/kg) 1 20.0 10.0 40% (40 mL) 60% (60 mL)  589 2 14.3 7.1 29% (40 mL) 71% (100 mL) 512 3 18.7 8.3 17% (20 mL) 83% (100 mL) 604

A Unit dosage form composition of Table 1 was reconstituted with 10 mL of Sterile WFI or USP Normal Saline then added into 100 mL 5% Dextrose Injection (D5W) or 0.9% Sodium Chloride (NS) bags and the osmolality of the resulting bag solution was determined as shown in Table 3b.

Example 4a Sodium Chloride in CXA-101Compositions

A. Improvement in the Purity of the Ceftolozane in CXA-101 Pharmaceutical Compositions with Varying Amounts of Sodium Chloride

A stability study was carried out at 30° C. and 60° C. and analyzed by HPLC. The sodium chloride content in the CXA-101 compositions is described in Table 4. The HPLC data are summarized in Tables 5-8. The data are also plotted in FIGS. 6-9 to show the trends of the purity, and the amounts of the composition peak 1, the composition with a RRT of 0.43 and the composition peak 3, and the composition peak 7 in the CXA-101 compositions with respect to NaCl.

TABLE 4 Sodium Chloride Content in the CXA-101 Compositions Samples NaCl content A1 481.0 mg NaCl per 1000 mg of ceftolozane A2 190.0 mg NaCl per 1000 mg of ceftolozane A3 125.0 mg NaCl per 1000 mg of ceftolozane A4 75.0 mg NaCl per 1000 mg of ceftolozane A5 50.0 mg NaCl per 1000 mg of ceftolozane

TABLE 5 Purity of Ceftolozane in CXA-101 Compositions with Varying Amounts of Sodium Chloride Day A A2 A3 A4 A5 t0/60° C. 0 96.6 98.0 97.9 97.8 97.7 t0/30° C. 0 98.1 97.8 97.8 97.7 1 day/60° C. 1 95.9 96.9 96.5 95.7 95.5 1 day/30° C. 1 98.2 97.7 97.7 97.6 3 days/60° C. 3 94.9 95.7 94.8 93.9 93.6 (Δ_(t0-t3)) (1.7) (2.3) (3.1) (3.9) (4.1) 3 day/30° C. 3 98.0 97.5 97.5 97.3 7 days/60° C. 7 93.6 94.0 94.2 92.3 91.9 7 day/30° C. 7 97.8 97.2 97.1 97.0 Total Δ/60° C. 3.07 4.06 3.7 5.48 5.83 Total Δ/30° C. 0.3 0.6 0.7 0.7

TABLE 6 HPLC Peak Area of Composition Peak 1 in CXA-101 Compositions with Varying Amounts of Sodium Chloride Day A1 A2 A3 A4 A5 t0/60° C. 0 0.95 0.31 0.3 0.36 0.39 t0/30° C. 0 0.47 0.36 0.36 0.39 1 day/60° C. 1 1.36 0.86 0.94 1.36 1.39 1 day/30° C. 1 0.48 0.40 0.42 0.48 3 days/60° C. 3 1.71 1.31 1.73 2.06 2.1 3 day/30° C. 3 0.53 0.50 0.52 0.58 7 days/60° C. 7 2.26 2.14 2.07 2.86 2.93 7 day/30° C. 7 0.62 0.63 0.66 0.72 INCREASE %/60° C. 1.31 1.83 1.77 2.5 2.54 INCREASE %/30° C. 0.15 0.27 0.30 0.33

TABLE 7 HPLC Peak Area of the Composition with a RRT of 0.43 and Composition Peak 3 in CXA-101 Compositions with Varying Amounts of Sodium Chloride Day A1 A2 A3 A4 A5 t0/60° C. 0 0.28 0.10 0.09 0.10 0.11 t0/30° C. 0 0.15 0.10 0.10 0.11 1 day/60° C. 1 0.37 0.13 0.16 0.35 0.36 1 day/30° C. 1 0.13 0.09 0.09 0.10 3 days/60° C. 3 0.68 0.21 0.31 0.71 0.71 3 day/30° C. 3 0.17 0.13 0.13 0.14 7 days/60° C. 7 1.04 0.36 0.30 0.81 0.81 7 day/30° C. 7 0.19 0.16 0.16 0.17 INCREASE %/60° C. 0.76 0.26 0.21 0.71 0.7 INCREASE %/30° C. 0.04 0.06 0.06 0.06

TABLE 8 The HPLC Peak Area of Composition Peak 7 in CXA-101 Compositions with Varying Amounts of Sodium Chloride Day A1 A2 A3 A4 A5 t0/60° C. 0 1.31 0.95 0.96 1.01 1.02 t0/30° C. 0 0.69 1.00 1.01 1.02 1 day/60° C. 1 1.37 1.10 1.10 1.23 1.29 1 day/30° C. 1 0.68 0.99 1.01 1.02 3 days/60° C. 3 1.43 1.19 1.27 1.41 1.46 3 day/30° C. 3 0.68 1.03 1.01 1.05 7 days/60° C. 7 1.49 1.31 1.35 1.55 1.57 7 day/30° C. 7 0.68 1.01 1.03 1.07 INCREASE %/60° C. 0.18 0.36 0.39 0.54 0.55 INCREASE %/30° C. NC 0.01 0.02 0.05

Conclusion:

The stability test demonstrates that high sodium chloride content enhances stability of CXA-101 Compositions.

The HPLC measurements on day 3 were used to analyze the stability of the CXA-101 compositions.

CXA-101 compositions comprising high amounts of sodium chloride (e.g., 125-1000 mg sodium chloride per 1000 mg of ceftolozane) were found to be more chemically stable than CXA-101 compositions comprising low amounts of sodium chloride (e.g., less than 125 mg sodium chloride per 1000 mg of ceftolozane). Table 5 shows that, by day 3 of heating at 60° C., sample A1, which has the highest salt concentration, is most stable, i.e., has the lowest A_(t0-t3) of all samples. By day 3, the sample with the lowest salt concentration, A5, has the highest A_(t0-t3) indicating the most degradation. Overall, A5 has degraded 141% more than A1. Further, Table 5 shows that, by day 3 of heating at 60° C., sample A3, which contains a lower salt concentration within the limits of the invention at 125 mg, is still significantly more stable than A4, a composition containing 75.0 mg of the salt. A3 has a A_(t0-t3) of 3.1, while A4 has a A_(t0-t3) of 3.9, meaning that A4 has degraded 26% more than A3.

B. Long-term Stability Study of CXA-101 Pharmaceutical Compositions with Varying Amounts of Sodium Chloride

Another stability study was carried out at 5° C. and 25° C. The sodium chloride content in the CXA-101 compositions is described in Table 9. The amounts of citric acid and L-arginine in each composition were the same. These samples were in lyophilized form and were placed on long-term (24-36 months), real time stability programs.

The composition peak 1 is considered “diagnostic” for formulation failure because it is the first peak to go out of trend or specification (1.5%). Thus, the stability of these CXA-101 compositions was also measured by the length of storage until formulation failure as indicated by the composition peak 1. The data in Table 9 were extrapolated from the data collected after 4 months. Clearly, based on the amount of the composition peak 1 in the compositions, the composition with about 480 mg sodium chloride per 1 gram ceftolozane active was significantly more stable than the compositions containing 125 mg or 62.5 mg sodium chloride per 1 gram of active ceftolozane (i.e., stability of ceftolozane compositions: 480>>125 mg>62.5 mg).

TABLE 9 The Peak 1 Failure Points of CXA-101 Compositions with Varying Amounts of Sodium Chloride Ceftolozane Peak 1 failure Peak 1 failure active, 1 g+ point at 5° C. point at 25° C. 480 mg NaCl 245 months  15 months  125 mg NaCl 70 months 5 months 62.5 mg NaCl  25 months  3 months* *Results at 3 months = 1.34%, 4 months = 1.15%

Example 4b Sodium Chloride in CXA-201 Compositions

A. Reduction of the Composition at RT=63 minutes in CXA-201Compositions

A stability study was carried out at 25° C. and analyzed by HPLC. CXA-201 compositions comprise ceftolozane and tazobactam, further comprising high, mid, or low amounts of sodium chloride (480, 125, or 62.5 mg NaCl per 1000 mg of ceftolozane, respectively). Comparison of the compositions are listed in Table 10. The amounts of the composition RT 63′, as measured by the HPLC method, are summarized in Table 11.

TABLE 10 Comparison of the CXA-201 Compositions Lot CXA-101 NaCl Tazobactam C1 10% High Na C2 20% Mid Na C3 20% Low Na C4 20% Mid Arginate C5 20% Low Arginate

TABLE 11 RT 63′ Peak Area at t = 3 months, 25° C./60% RH storage 1^(st) data 2nd data 3rd data collection collection collection Area Area Area Sample Summary RT % RT % RT % C1 High salt + 63.90 0.03 63.30 0.08 62.49 0.14 Tazo Na C2 Mid salt + 63.78 0.06 63.12 0.12 62.45 0.28 Tazo Na C3 Low salt + 63.75 0.12 63.11 0.14 62.46 0.29 Tazo Na C4 Mid salt + 63.76 0.10 63.16 0.13 62.44 0.28 Tazo Arg C5 Low salt + 63.72 0.08 63.14 0.16 62.46 0.33 Tazo Arg

Conclusion:

At the three month time point, the reduced salt formulations were observed to be not as stable as the full salt formulation; and trends indicate that reduction in salt causes at least 1.5-fold greater composition at RT=63 minutes, as measured by HPLC. The compositions comprising 480 mg NaCl per 1000 mg of ceftolozane had the least amount of the composition RT 63′ after 3 months at 25° C. The amount of the composition RT 63′ in the compositions comprising 125 mg NaCl per 1000 mg of ceftolozane was 1.5-fold or greater than the amount of the composition 63′ in the compositions comprising 480 mg NaCl per 1000 mg of ceftolozane. The amount of the composition RT 63′ in the compositions comprising 62.5 mg NaCl per 1000 mg of ceftolozane was 2-fold or greater than the amount of the composition RT 63′ in the compositions comprising 480 mg NaCl per 1000 mg of ceftolozane. Thus, the CXA-201 compositions comprising high amounts of sodium chloride (e.g., 125-1000 mg sodium chloride per 1000 mg of ceftolozane) were more chemically stable than the compositions comprising low amounts of sodium chloride (e.g., less than 125 mg sodium chloride per 1000 mg of ceftolozane).

B. Improvement in the Purity of Ceftolozane in CXA-201 Pharmaceutical Compositions with Varying Amounts of Sodium Chloride

A stability study was carried out at 30° C. and 60° C. analyzed by HPLC. The sodium chloride content in the CXA-201 compositions is described in Table 12. The HPLC data at 30 and 60° C. are summarized in Tables 13-16. The data are also plotted in FIGS. 6-9 to show the trends of the purity, and the amounts of the composition peak 1, the composition with a RRT of 0.43 and the composition peak 3, and the composition peak 7 in the CXA-201 compositions with respect to NaCl.

TABLE 12 The Sodium Chloride Content in the CXA-201 Compositions Samples NaCl content B1 481.0 mg sodium chloride per 1000 mg of ceftolozane B2 125.0 mg sodium chloride per 1000 mg of ceftolozane B3 75.0 mg sodium chloride per 1000 mg of ceftolozane B4 50.0 mg sodium chloride per 1000 mg of ceftolozane

TABLE 13 The Purity of Ceftolozane in CXA-201 Compositions with Varying Amounts of Sodium Chloride Day B1 B2 B3 B4 t0 0 98.1 97.8 97.8 97.7 1 day/60° C. 1 97.2 96.3 96.2 96.0 1 day/30° C. 1 98.2 97.7 97.6 97.6 3 days/60° C. 3 95.4 (2.7) 94.9 (2.9) 94.7 (3.1) 94.6 (3.1) (Δ_(t0-t3)) 3 day/30° C. 3 98.0 97.5 97.4 97.3 7 days/60° C. 7 92.7 93.8 93.6 93.4 7 day/30° C. 7 97.8 97.2 97.0 96.9 Total Δ/60° C. 5.3 4.0 4.2 4.3 Total Δ/30° C. 0.3 0.6 0.8 0.8

TABLE 14 The HPLC Peak Area of Composition Peak 1 in CXA-201 Compositions with Varying Amounts of Sodium Chloride Day B1 B2 B3 B4 t0 0 0.47 0.38 0.38 0.41 1 day/60° C. 1 1 1.08 1.09 1.14 1 day/30° C. 1 0.48 0.44 0.45 0.49 3 days/60° C. 3 1.85 1.64 1.66 1.71 3 day/30° C. 3 0.53 0.53 0.56 0.61 7 days/60° C. 7 3.3 2.28 2.25 2.29 7 day/30° C. 7 0.62 0.67 0.71 0.77 INCREASE %/60° C. 2.83 1.9 1.87 1.88 INCREASE %/30° C. 0.15 0.29 0.33 0.36

TABLE 15 The Total HPLC Peak Area of the Composition with a RRT of 0.43 and Composition Peak 3 in CXA-201 Compositions with Varying Amounts of Sodium Chloride Day B1 B2 B3 B4 t0 0 0.15 0.12 0.12 0.12 1 day/60° C. 1 0.36 0.35 0.31 0.32 1 day/30° C. 1 0.13 0.12 0.13 0.12 3 days/60° C. 3 0.92 0.67 0.65 0.62 3 days/30° C. 3 0.17 0.16 0.17 0.16 7 days/60° C. 7 1.29 0.78 0.75 0.71 7 days/30° C. 7 0.19 0.19 0.20 0.20 INCREASE %/60° C. 1.14 0.66 0.63 0.59 INCREASE %/30° C. 0.04 0.07 0.08 0.08

TABLE 16 The HPLC Peak Area of Composition Peak 7 in CXA-201 Compositions with Varying Amounts of Sodium Chloride Day B1 B2 B3 B4 t0 0 0.69 1.01 1.01 1.01 1 day/60° C. 1 0.73 1.12 1.15 1.18 1 day/30° C. 1 0.68 1.00 0.99 0.95 3 days/60° C. 3 0.8 1.24 1.27 1.27 3 days/30° C. 3 0.68 1.00 1.01 1.03 7 days/60° C. 7 0.94 1.32 1.35 1.4 7 days/30° C. 7 0.68 1.02 1.05 1.06 INCREASE %/60° C. 0.25 0.31 0.34 0.39 INCREASE %/30° C. NC 0.01 0.04 0.05

Conclusion:

The stability data shows that high sodium chloride content enhances stability of CXA-201 compositions.

Similarly to CXA-101 compositions, CXA-201 compositions comprising high amounts of sodium chloride (e.g., 125-1000 mg sodium chloride per 1000 mg of ceftolozane) were found to be more chemically stable than CXA-201 compositions comprising low amounts of sodium chloride (e.g., less than 125 mg sodium chloride per 1000 mg of ceftolozane). Table 10 shows that, by day 3 of heating at 60° C., sample B1 containing the highest salt concentration is most stable, i.e., has the lowest A of all samples. By day 3, the sample with the lowest salt concentration, B4, has the highest Δ_(t0-t3) indicating the most degradation. Overall, B4 has degraded 15% more than B1.

Example 5 HPLC Analysis of Compositions Comprising Ceftolozane

The purity of the ceftolozane in the pharmaceutical compositions was measured using the analytical HPLC method described below.

The stability studies and HPLC methodologies described herein were used to acquire the data provided in the following examples.

Analytical HPLC Method A. Operative Conditions

Column Develosil ODS-UG-5; 5 μm, 250 × 4.6 mm (Nomura Chemical, Japan) Mobile phase Sodium perchlorate buffer solution (PH 2.5)/CH₃CN 90:10 (v/v) Flow rate 1.0 mL/min Wavelength 254 nm Injection volume 10 μL Oven Temperature 45° C. Run Time 85 minutes

TABLE 17 Gradient Profile used in analytical HPLC method: Time (min) A % B % 0 75 25 30 70 30 60 0 100 85 0 100 85.1 75 25 110 75 25

B. Mobile Phase Preparation.

Sodium perchlorate buffer solution was made by dissolving 14.05 g of sodium perchlorate monohydrate in 1000.0 mL of water followed by adjusting pH to 2.5 with diluted perchloric acid (1 in 20).

Mobile phase was then made by mixing sodium perchlorate buffer solution (pH 2.5) and acetonitrile in the ratio 90:10 (v/v).

Sodium acetate buffer solution pH 5.5 (diluent) was made by dissolving 1.36 g of sodium acetate trihydrate in 1000.0 mL of water followed by adjusting to pH 5.5 with diluted acetic acid (1 in 10).

C. Sample Preparation.

Sample solution: dissolve 20.0 mg, exactly weighed, of the Sample, in 20.0 mL of water (Prepare just before injection into HPLC system).

System suitability solution (1%): take 1.0 mL of the sample solution (the first sample if more are present) and transfer into a 100.0 mL volumetric flask, dilute with water to volume and mix.

D. HPLC Analysis Procedure

1. Inject blank (water) 2. Inject system suitability solution and check for tailing factor and theoretical plate number for the CXA-101 peak:

-   -   The tailing factor must not be greater than 1.5     -   Theoretical plates number must not be less than 10000         3. Inject sample solution         4. Inject system suitability solution and check for tailing         factor and theoretical plate number for the CXA-101 peak.     -   The tailing factor must not be greater than 1.5     -   Theoretical plates number must not be less than 10000         5. Identify the peaks of related substances in the sample         chromatogram based on the reference chromatogram reported in         FIG. 1 or, alternatively, on the basis of the following RRT         values listed in Table 18.

TABLE 18 Identities and RRTs of the Products Related to Ceftolozane Compound RRT Proposed Structure Source Peak 1 3-side chain ~0.14

Degradation product and process product Peak 2 ~0.16 Unidentified Process product Peak 3 ~0.4 Unidentified Process product Peak 4 ~0.6 Unidentified Process product Peak 5 7-Epimer type ~0.9

Degradation product and process product Peak 6 ~1.1 NA Process product Peak 7 Δ3 Isomer type ~1.30

Degradation product and process product Peak 8 ~1.37 Unidentified Process product Peak 9 Anti-Isomer type ~1.7

Process product and Degradation product Peaks 10, 11 ~2.3 Unidentified Process product

E. Calculations

I. Report for each related substance its amount as expressed by area percent.

$C_{i} = \frac{A_{i} \times 100}{A_{t} + {\sum A_{i}}}$

-   -   wherein:     -   C_(i)=Amount of related substance i in the sample, area %     -   A_(i)=Peak area of related substance i in the sample         chromatogram     -   A_(t)=Area of CXA-101 peak in the sample chromatogram     -   A_(t)+ΣA_(i)=Total peaks area in the sample chromatogram     -   Consider as any unspecified compound, each peak in the         chromatogram except CXA-101, peaks from 1 to 11 and every peak         present in the blank chromatogram and report the largest.

II. Report the total composition content as expressed by the following formula:

$C_{T} = \frac{A_{i} \times 100}{A_{t} + {\sum A_{i}}}$

-   -   wherein:     -   C_(T)=total composition content in the sample, area %     -   A_(t)=area of CXA-101 peak in the sample chromatogram     -   ΣA_(i)=total peak areas of compositions in the sample         chromatogram

Example 6 Ceftolozane without the Presence of a Stabilizing Agent

Five samples were prepared, the components of which are shown in Table 19 below. Each sample contained 1000 mg of ceftolozane active, 40 mg citric acid monohydrate (equivalent of 36 mg citric acid anhydrous), and the same amount of L-arginine. Stabilizing reagents in four samples are 480 mg sodium chloride, 300 mg of trehalose, 300 mg of sucrose, and 300 mg of PVP, respectively. One sample was a control that contained no stabilizing reagent. The samples were in lyophilized form and stored at 60° C. for 7 days. The purities of the samples were monitored by HPLC on day 0, day 1, day 3 and day 7.

TABLE 19 Comparison between stabilizing reagents Excipient Treha- Su- NaCl lose crose PVP None Ceftolozane 1000 1000 1000 1000 1000 amount Excipient 480 300 300 300 N/A amount Purity: t₀ 98.42 98.09 98.14 97.89 97.94 60° C./1 d 97.85 96.73 96.97 96.05 96.15 60° C./3 d 97.21 95.36 95.81 94.57 94.53 60° C./7 d 95.65 94.21 94.19 92.78 92.06 Purity Δ −2.77% −3.88% −3.95% −5.11% −5.88% (0-7 d)

As shown in Table 19, the sample containing sodium chloride exhibited the best stability. The purity of ceftolozane in the sample containing sodium chloride had the slightest purity drop over 7 days. This experiment further supports the discovery that sodium chloride provides surprisingly better stabilizing effect than the other reagents.

The purity of the ceftolozane in a composition comprising 100 mg ceftolozane was also evaluated with no stabilizing agent after 3 days at 70° C. by measuring the residual rate. The residual rate is measured by detecting the amount of ceftolozane in a sample before and after a stability test using HPLC, and determining the percentage of ceftolozane lost during the stability test. The residual rate of ceftolozane without any stabilizing agent (i.e., 100 mg of ceftolozane) after 3 days at 70° C. was 51.2%, meaning that the HPLC peak area after the stability test for ceftolozane (i.e., 3 days at 70 degrees C.) was about 51.2% of the HPLC peak area for ceftolozane at the start of the stability test (i.e, at time zero considered to be 100%).

As shown in Table 19, ceftolozane without without any stabilizing agent exhibited an overall decrease in purity of 5.88%. This experiment further supports the need for an additive with a stabilizing effect on ceftolozane.

Example 7 Manufacturing Procedure of a Lyophilized Pharmaceutical Composition Comprising Ceftolozane and Sodium Chloride (CXA-101)

There are four main steps in the manufacture of a CXA-101 pharmaceutical composition: dissolution, sterile filtration, bulk lyophilization, and packaging into Sterbags®. These four main steps are composed of a total of 20 minor steps. The flowchart of the manufacturing process is described below.

I. Dissolution

1. A prescribed amount of WFI (e.g., 81 kg WFI) is charged into a dissolution reactor. 2. A prescribed amount of citric acid (e.g., 20.7 mg anhydrous citric acid per 1000 mg ceftolozane active) is added. 3. The solution is cooled to 5° C. to 10° C. 4. A prescribed amount of CXA-101 drug substance (e.g., 9.462 kg active) is added to the solution. 5. A prescribed amount of L-arginine (e.g., 587 mg L-arginine per 1000 mg ceftolozane active) is slowly added to the solution. 6. A check for complete dissolution is performed. Solution pH is verified to be in the target range of 6.5 to 7.0. 7. A prescribed amount of sodium chloride (e.g., 476 mg sodium chloride per 1000 mg ceftolozane active) is added to the solution. 8. A check for complete dissolution is performed. Solution pH is verified to be in the target range of 6.0 to 7.0. If the pH is out of this range, adjust with either L-Arginine or citric acid. 9. WFI is added to bring the net weight to 124.4 kg and the solution is mixed well. 10. Samples are withdrawn for testing of final pH. II. Sterile filtration 11. The solution is passed through a filter (pore size 0.45 μm) followed by two more filters (pore size 0.22 μm) onto a shelf on the Criofarma lyophilizer. 12. The line is washed with WFI. 13. The washing solution from Step 12 is passed through sterile filtration. III. Bulk lyophilization 14. The washing solution is loaded onto a separate shelf on the lyophilizer (and later discarded). 15. The solution is lyophilized until dry. 16. The product shelf is cooled to 20° C.±5° C. IV. Packaging into Sterbags® 17. The lyophilized pharmaceutical composition is milled. 18. The milled powder is sieved. 19. The sieved powder is blended for 30 minutes. 20. The powder is then discharged into Sterbags®

Example 8 Efficacy Testing of CXA-201

Ceftolozane/tazobactam, an antibiotic candidate being developed to treat certain Gram-negative infections, includes ceftolozane, a cephalosporin that has demonstrated more potent in vitro activity against Pseudomonas aeruginosa as compared to the currently available cephalosporins, with tazobactam, a Beta-lactamase inhibitor. The addition of tazobactam broadens coverage to include most Extended-spectrum B-lactamase (ESBL)-producing Escherichia coli (E. coli), Klebsiella pneumoniae, and other Enterobacteriaceae.

Ceftolozane/tazobactam is being developed for the potential treatment of Complicated Urinary Tract Infections (cUTI) and Complicated Intra-Abdominal Infections (cIAI). In pivotal Phase 3 clinical trials of ceftolozane/tazobactam in cUTI when studied against levofloxacin and of ceftolozane/tazobactam, in combination with metronidazole, in cIAI when studied against meropenen, ceftolozane/tazobactam met its primary endpoints of statistical non-inferiority. Ceftolozane/tazobactam is also being developed for the potential treatment of Hospital-Acquired Bacterial Pneumonia (HABP)/Ventilator-Associated Bacterial Pneumonia (VABP) at a dose of 3 g every 8 hours.

CXA201 has strong activity against the most common and problematic Gram-negative pathogens. The tables below show Per Pathogen Micro Eradication (ME Population) from treatment of urinary tract infections in Phase III clinical trials and the per pathogen response rates for treatment of complicated intra-abdominal infections in Phase III clinical trials.

TABLE 20 Per Pathogen Micro Eradication (ME Population) from treatment of urinary tract infections in Phase III clinical trials CXA-201 treating cUTI Gram (−) Aerobes 287/323 (88.9%) Enterobacteriaceae 281/316 (88.9%) E. coli 237/262 (91%) ESBL 27/36 (75%) CTXM14/15 20/27 (74%) K. pneumoniae 21/25 (84%) ESBL 7/10 (70%) CTXM14/15 5/8 (62.5%) P. aeruginosa 6/7 (85.7%)

TABLE 21 Per pathogen response rates for treatment of complicated intra-abdominal infections in Phase III clinical trials CXA-201 for cAIA (with metronidazole) Gram (−) Aerobes 234/243 (96.3) Enterobacteriaceae ESBL 22/22 (100) CTXM14/15 12/12 (100) E. coli 193/201 (96.0) ESBL 14/14 (100) CTXM14/15 9/9 (100) K. pneumoniae 28/28 (100) ESBL 6/6 (100) CTXM14/15 4/4 (100) P. aeruginosa 25/25 (100) AmpC 4/4 (100) Surveillance data from a large 2011 US surveillance study are summarized in Table 22 below. Ceftolozane/tazobactam demonstrated higher in vitro activity than currently available cephalosporins and piperacillin/tazobactam when tested against Enterobacteriaceae (Table 22). Ceftolozane/tazobactam was the most active β-lactam agent tested against P. aeruginosa and was 2 to 8-fold more active than ceftazidime or cefepime. Similar to other β-lactam compounds tested, ceftolozane/tazobactam exhibited modest activity against Acinetobacter spp. This data also demonstrated that, with the exception of K. pneumoniae with an ESBL phenotype, ceftolozane-tazobactam inhibited >90% of Enterobacteriaceae at MIC values <8 μg/mL. Notably, 94.6% of E. coli with an ESBL phenotype were inhibited at <8 μg/mL of ceftolozane/tazobactam.

TABLE 22 Activity of Ceftolozane and Ceftolozane/Tazobactam with that of other β-Lactams and a β-Lactam-Inhibitor Combination against Recent Gram-Negative US Surveillance Isolates (2011) MIC

/MIC

 (μg/mL) No. of Ceftolozane/ Piperacillia- Species isolates Ceftolozane Tazobactam Ceftazidine Cefepinse Tazobactam Aztreonam E. coli (non-ESBL) 1076 0.25/0.25 0.12/0.25 0.12/0.25 ≦0.5/≦0.5 2/4 ≦0.12/≦0.12 K. pneumoniae (non-ESBL) 551 0.25/0.5  0.25/0.5  0.12/0.5  ≦0.5/≦0.5 4/8 ≦0.12/≦0.12 P. mirabilis 203 0.5/0.5 0.5/0.5 0.06/0.06 ≦0.5/≦0.5 ≦0.5/1     ≦0.12/≦0.12 Enterobactor spp. 525 0.5/16  0.25/8   0.25/>32  ≦0.5/2      2/64 ≦0.12/>16  Serratia spp. 287 0.5/1  0.5/1  0.12/0.5  ≦0.5/≦0.5 2/4 ≦0.12/0.5    Citrobacter spp. 174 0.25/16  0.25/8   0.25/>32  ≦0.5/1      2/64 ≦0.12/>16  P. aeringinosa 973 1/4 1/4  2/32  4/16  8/>64  8/>16 Acinetobacter spp. 208  16/>32  8/>32  32/>32  16/>16 >64/>64 >16/>16 H. influenzae 51 0.12/0.25 0.12/0.12 0.06/0.12 ≦0.5/≦0.5 ≦0.5/≦0.5 ≦0.12/≦0.12 Ceftolozane/tazobactam M

 determined at a fixed concentration of 4 μg/mL tazobactam

indicates data missing or illegible when filed 

1. A method of treating an infection selected from the group consisting of Complicated Urinary Tract Infections (cUTI), Complicated Intra-Abdominal Infections (cIAI) and Hospital-Acquired Bacterial Pneumonia (HABP)/Ventilator-Associated Bacterial Pneumonia (VABP), the method comprising a. reconstituting a lyophilized unit dosage form composition comprising the following components Nominal Composition Component mg per 1 g ceftolozane active Ceftolozane Sulfate about 1147 (providing 1,000 mg ceftolozane active) Citric Acid 21 Sodium Chloride 450-500 L-Arginine 500-700 Tazobactam about 537 (providing 500 mg tazobactam free acid Sodium per 1,000 mg ceftolozane active)

in one or more pharmaceutically acceptable liquid carriers selected from the group consisting of 0.9% sodium chloride aqueous solution for injection, 0.45% sodium chloride aqueous solution for injection, and 5% dextrose for injection and water for injection, to form a dissolved unit dosage form; b. diluting the dissolved unit dosage form in one or more pharmaceutically acceptable liquid carriers selected from the group consisting of 0.9% sodium chloride aqueous solution for injection, 0.45% sodium chloride aqueous solution for injection, and 5% dextrose for injection and water for injection, to form a parenteral pharmaceutical formulation comprising a therapeutically effective amount of ceftolozane sulfate and tazobactam, and having an osmolality of about 400-600 mOsm/kg and a pH of about 5-7; and c. intravenously administering to a patient in need thereof the parenteral pharmaceutical formulation comprising the therapeutically effective dose of ceftolozane sulfate and tazobactam; tazobactam in an amount providing 500 mg of tazobactam acid per 1,000 mg of ceftolozane active in the parenteral pharmaceutical formulation; 450 to 500 mg of sodium chloride per 1,000 mg of ceftolozane active in the parenteral pharmaceutical formulation; and L-arginine.
 2. The method of claim 1, further comprising intravenously administering the parenteral pharmaceutical formulation over a period of about 1 hour to the patient.
 3. The method of claim 1, further comprising administering a second and a third parenteral pharmaceutical formulation comprising ceftolozane and tazobactam to a patient in need thereof by repeating steps (a)-(c) three times a day.
 4. The method of claim 1, wherein the infection is a Complicated Urinary Tract Infections (cUTI) or Complicated Intra-Abdominal Infections (cIAI).
 5. The method of claim 4, wherein the parenteral pharmaceutical formulation contains a total of 1,000 mg of ceftolozane active present as ceftolozane sulfate.
 6. The method of claim 5, wherein the parenteral pharmaceutical formulation contains a total of 500 mg of tazobactam acid present as tazobactam sodium.
 7. The method of claim 1, wherein the lyophilized unit dosage form composition comprises 1,000 mg of ceftolozane active present as ceftolozane sulfate.
 8. The method of claim 1, wherein the lyophilized unit dosage form composition is obtained by a process comprising the steps of: a. lyophilizing a first aqueous solution in the absence of tazobactam, the first aqueous solution comprising ceftolozane, 450 mg to 500 mg of sodium chloride per 1,000 mg of ceftolozane active, at a pH of 6-7 prior to lyophilization to obtain a first lyophilized ceftolozane composition, b. lyophilizing a second solution comprising tazobactam in the absence of ceftolozane to form a second lyophilized tazobactam composition; and c. blending the first lyophilized ceftolozane composition and the second lyophilized tazobactam composition to obtain the antibacterial composition; and d. packaging a portion of the antibacterial composition as the lyophilized unit dosage form composition.
 9. The method of claim 8, wherein the first aqueous solution further comprises L-arginine in an amount providing a pH of about 6-7.
 10. The method of claim 8, wherein the first aqueous solution comprises water and the following components: Nominal Composition Component mg per Vial Ceftolozane Sulfate about 1147 (providing 1,000 mg ceftolozane active) Citric Acid  21 Sodium Chloride 487 L-Arginine 600 Q.S. for pH adjustment Nitrogen Q.S.


11. A method of treating a bacterial infection in a subject comprising administering to a patient in need thereof a parenteral pharmaceutical antibiotic composition having an osmolality of 400-500 mOsm/kg at a pH of 5-7, the composition comprising a. an amount of ceftolozane sulfate providing 1,000 mg ceftolozane active, b. about 450 mg to 500 mg sodium chloride per 1,000 mg ceftolozane active, and c. an amount of tazobactam sodium providing 500 mg of tazobactam free acid; in a pharmaceutically acceptable liquid carrier selected from the group consisting of 0.9% sodium chloride aqueous solution for injection, 5% dextrose for injection and water for injection.
 12. The method of claim 11, wherein the bacterial infection is a complicated urinary tract infection.
 13. The method of claim 11, wherein the bacterial infection is a complicated intra-abdominal infection.
 14. The method of claim 11, wherein the bacterial infection comprises a bacteria selected from the group consisting of: Staphylococcus aureus, Escherichia coli, Acinetobacter baumanii, Haemophilus influenzae, Klebsiella pneumonia, and Pseudomonas aeruginosa.
 15. The method of claim 11, further comprising the steps of a. reconstituting a unit dosage form of ceftolozane/tazobactam having the composition: Nominal Composition Component mg per Vial Ceftolozane Sulfate about 1147 (providing 1,000 mg ceftolozane active) Citric Acid  21 Sodium Chloride 487 L-Arginine 600 Q.S. for pH adjustment Tazobactam about 537 (providing 500 mg tazobactam free acid Sodium per 1,000 mg ceftolozane active) Nitrogen Q.S.

in a pharmaceutically acceptable liquid carrier to obtain a reconstituted antibiotic composition; b. intravenously administering the reconstituted antibiotic composition within the parenteral pharmaceutical antibiotic composition.
 16. The method of claim 15, further comprising repeatedly intravenously administering separate doses of the parenteral pharmaceutical antibiotic composition to a patient in need thereof three times per day.
 17. The method of claim 16, wherein each separate parenteral pharmaceutical antibiotic compositions are administered to the patient over a 1 hour period. 